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PubMed2006

An ITS-RFLP method to identify black Aspergillus isolates responsible for OTA contamination in grapes and wine.

Ochratoxigenic mycobiota in grapes from representative wine regions in Valencia was identified. Black aspergilli were predominant among the different Aspergillus spp. isolated. Restriction digestion analysis of the ITS products was tested as a rapid method to identify isolates of black Aspergillus species from grapes. Restriction endonuclease digestion of the ITS products using the endonucleases HhaI, NlaIII and RsaI, distinguished five types of restriction fragment length polymorphism (RFLP) corresponding to Aspergillus niger, Aspergillus tubingensis, Aspergillus carbonarius and Aspergillus aculeatus species. In addition, a new RFLP type in the A. niger aggregate was identified. The fragments obtained by digestion with the endonuclease NlaIII could be used to identify these new isolates. Black Aspergillus isolates were tested for their ability to produce OTA. Most of the isolates that produced ochratoxin A in YES medium belonged to A. carbonarius species. These results support evidence that A. carbonarious greatly contributes to OTA contamination in grapes and consequently in wine. The ITS-RFLP assay is proposed as a rapid and easy method to identify black Aspergillus species isolated from grapes, especially in studies that involve a large number of isolates.

Aspergillus↗
PubMed2006

Ochratoxin A and ochratoxigenic Aspergillus species in Argentinean wine grapes cultivated under organic and non-organic systems.

The evolution of contamination with Aspergillus section Nigri and ochratoxin A occurrence was evaluated in four vineyards located at Mendoza province, Argentina during 2003-2004. The survey included two grape varieties, one of late maturation (Bonarda) and the other of early maturation (Tempranillo). The vineyards were set under non-organic and organic cropping systems. Bunches of grapes at different growth stages were collected, and berries (50 by sample) were plated on Petri dishes containing Dichloran 18% Glycerol Agar (DG18) and Dichloran Rose Bengal Chloramphenicol Agar (DRBC) media. After an incubation period of 7 days at 25 degrees C+/-1 degrees C, the mycoflora belonging to Aspergillus section Nigri was identified. The ability to produce ochratoxin A (OTA) by the potential ochratoxigenic species was evaluated on YES (2% yeast extract, 15% sucrose) medium. The cultures were incubated at 30 degrees C+/-1 degrees C for 10 days in darkness. The OTA content of the grapes was determined by HPLC. Through the different growth stages, from setting to harvest, grape contamination by the Aspergillus species, section Nigri increased. The main species isolated belonged to the A. niger aggregate. From 246 strains evaluated 24% was ochratoxigenic. OTA was not detected in grapes during the survey.

Agriculture↗
PubMed2006

Occurrence of pathogenic fungal species in Tunisian vineyards.

Mycotoxins are secondary metabolites produced by filamentous fungi detected in food, such are grapes. OTA was evaluated in ten handle musts from different Tunisian vineyard. This mycotoxin was found at levels 1.1 mug/L to 4.3 mug/L. A survey was conducted to assess the contamination of the Tunisian vineyard with pathogenic fungal species, in particular those responsible of the OTA production. The results were evaluated for the first time in parcels cultivated in the North, in the Centre and in the South of the country. Italia Muscate and Superior Seedless varieties were concerned at three developmental stages of the berry, setting, veraison and maturity. Carigon variety was used as positive control for musts contaminating by OTA. The main fungal species isolated were Aspergillus spp. (33.32%), Botrytis cinerea (23.32%), Alternaria spp. (12.80%), Cladosporium spp. (10.59%) and Penicillium spp. (8.3%). The isolates of the Aspergillus genus were identified as Aspergillus niger aggregate (77%), Aspergillus carbonarius (15%) and Aspergillus flavus (8%). Their presence was characterized by a significant decrease in the Centre during the veraison and a slight increase in the North and the South during the maturity stage. Furthermore, when comparing Superior Seedless and Italia Muscate cultivated in the same area, the aspergilli were particularly less abundant at the setting stage in the case of Superior Seedless. There is no correlation between the OTA amount in musts and the contamination by Aspergillus species in different vineyards and for grape varieties studied.

Alternaria↗
PubMed2006

Studies on the interaction between grape-associated filamentous fungi on a synthetic medium.

Eleven fungi isolated from grapes and sun-dried grapes (Alternaria alternata, Cladosporium herbarum, Eurotium amstelodami, Penicillium janthinellum, P. decumbens, Trichoderma harzianum, Candida sp., Aspergillus carbonarius OTA-negative, A. carbonarius OTA-positive, A. niger var. niger. and A. japonicus var. aculeatus), were grown in SNM medium at different water activities (0.82-0.97) and temperature (20-40 degrees C) levels for 18 days. Pairs of one Aspergillus faced with one non-Aspergillus were grown at 0.87-0.97 a(w) and at 20 and 30 degrees C. In single cultures, daily radii were recorded. In paired cultures radii were recorded and each A. section Nigri isolate was given a dominance score. At high temperatures and low water activities, Penicillium isolates, E. amstelodami and A. niger var. niger showed higher growth rates, while T. harzianum only grew well at the highest water activity. In addition, A. section Nigri was dominant in most paired assays, being only surpassed by T. harzianum at 0.97 a(w) and 20 degrees C. Thus, prevalence of A. section Nigri in sun-dried grapes can be explained by its adaptation to environmental conditions of sun-drying, and by its capability to dominate other fungal species when coming into contact with them.

Colony Count, Microbial↗
PubMed2006

Occurrence of ochratoxigenic fungi and ochratoxin A in grapes from a Tunisian vineyard.

The occurrence of ochratoxin A (OTA) and the identification of the ochratoxigenic microbiota in Tunisian grapes were studied for the first time. Black aspergilli were the dominant genus among the filamentous fungi isolated from grapes and were the only potential OTA-producing fungi found. The most abundant species were member of Aspergillus niger aggregate (63%) and Aspergillus carbonarius (36%). Uniseriate aspergilli were rarely present (1%). Of the A. carbonarius isolates, 97% were OTA positive but only 3% of the A. niger aggregate isolates were OTA positive. During grape maturation, the frequency of black aspergilli increased due to increase of the numbers of A. carbonarius. Musts (n=24) obtained from grapes collected at the different sampling times were analyzed for their OTA content. Up to 37% of the musts contained OTA at levels varying between 0.59 and 2.57 microg/l. The amounts of OTA in musts increased as grapes matured. These results indicate that A. carbonarius is the main cause of OTA contamination of Tunisian grapes.

Aspergillus↗
PubMed2006

Effect of water activity and temperature on growth of ochratoxigenic strains of Aspergillus carbonarius isolated from Argentinean dried vine fruits.

Aspergillus carbonarius is an ochratoxin A (OTA) producing fungus, predominantly responsible for the production of this mycotoxin in grapes, wine and dried vine fruits. The objective of this study was to determine the in vitro effects of water activity (a(w), 0.80-0.95) and temperature (15-35 degrees C) on lag phase extension and radial growth rate of a cocktail inoculum of four strains of A. carbonarius. The maximum growth rate was observed at 0.95 a(w) and 30 degrees C (17.46 mm day(-1)). In general, growth rates increased with the increment of a(w). No growth was observed at a(w) below 0.85. For all a(w) levels tested the highest growth rate was detected at 30 degrees C. At 15 degrees C growth only occurred at the higher a(w) levels evaluated (0.925 and 0.95) at a growth rate of 3.82 and 5.57 mm day(-1) respectively. The shortest lag phase (0.26 days) was found at 0.925 a(w) and 35 degrees C. At marginal conditions of a(w) and temperature the lag phases increased, being the highest registered at 20 degrees C and 0.89 a(w) (33.7 days). The pattern of effects of environmental factors on growth was similar among Argentinean A. carbonarius strains and those from several European countries, Israel and Australia.

Argentina↗
PubMed2006

Influence of carvacrol on survival of Botrytis cinerea inoculated in table grapes.

In this paper, the role of carvacrol vapour atmosphere on Botrytis cinerea inoculated in PDA or in grape berries was studied. Four concentrations inside packages were assayed (0.05, 0.2, 0.5 and 1.0 ml l(-1)). All concentrations inhibited totally the growth of B. cinerea in PDA, while in berries the reduction of decayed fruits was significantly greater as carvacrol concentration increased. In addition, the fungal growth (area and volume of infection) was also reduced and dependent on carvacrol concentration. Ethylene and respiration rate (berry physiological parameters) increased drastically in control inoculated-grapes, while these increases were lower as higher were the carvacrol applied doses. The data presented in this work suggest that carvacrol could be used as an innovative tool to control fungal decay during table grape storage, as alternative to the use of synthetic fungicides such as SO2.

Botrytis↗
PubMed2004

Resveratrol inhibits phenotypic and functional maturation of murine bone marrow-derived dendritic cells.

This study investigated the effects of resveratrol, a natural polyphenol found in grapes and grape products such as wine and having a wide range of biological and pharmacological activities effecting on the phenotypic and functional maturation of bone marrow (BM)-derived dendritic cells (DC). Resveratrol inhibited the expression of costimulatory molecules (CD80 and CD86), and major histocompatibility complex (MHC) classes I and II significantly, and had the same effect dose-dependently on DC. Resveratrol also significantly suppressed the ability of BM-DC to produce intracellular IL-12 p40/p70 and secretory IL-12 p70 in response to lipopolysaccharides (LPS) stimulation. Resveratrol-treated DC were highly efficient in antigen capture via mannose receptor-mediated endocytosis. Also, they were poor stimulators of naïve allogeneic T-cell proliferation and induced lower levels of IL-2 in responding T cells. These results indicate the immunosuppressive properties of resveratrol, which may be therapeutically useful in controlling chronic immune and/or inflammatory diseases through the down-regulation of DC differentiation and maturation.

Animals↗
PubMed2005

Proanthocyanidin from grape seeds potentiates anti-tumor activity of doxorubicin via immunomodulatory mechanism.

The purpose of this study was to investigate the anti-tumor activities of proanthocyanidin (PA) from grape seeds and doxorubicin (DOX) in vitro as well as in vivo, either alone or in combination and to explore the immunomodulatory mechanism in tumor-bearing mice. PA (12.5 approximately 200 mg/l) or DOX (0.01 approximately 1 mg/l) for 24 h significantly inhibited YAC-1 cell proliferation (IC(50) 57.53 or 0.198 mg/l, respectively) in a concentration-dependent manner using microculture tetrazolium (MTT) assay. Meanwhile, a combination of PA (12.5, 25 mg/l) with DOX strongly inhibited cell proliferation with IC(50) values of DOX decreasing by 0.09 and 0.045 mg/l, respectively. In mouse tumor xenograft models, intraperitoneal administrations of PA (10 mg/kg) daily or DOX (2 mg/kg) every other day for 9 days significantly inhibited the growth of sarcoma 180, whereas a combination of the two strongly inhibited tumor growth as compared with PA or DOX alone (p<0.01). In contrast to PA treatment, DOX inhibited Con A-stimulated lymphocyte proliferation, IL-2 and IFN-gamma productions, NK cell cytotoxicity and CD4+/CD8+ ratio, while the administration of PA combined with DOX significantly enhanced the above immune responses as compared with the tumor-bearing control (p<0.01). Taken together, these results suggest that PA has anti-tumor activity and increases the anti-tumor activity of DOX, and the mechanism might be related partially to immunopotentiating activities through the enhancements of lymphocyte proliferation, NK cell cytotoxicity, CD4+/CD8+ ratio, IL-2 and IFN-gamma productions.

Animals↗
PubMed2005

Effects of cyclosporine on oxidant/antioxidant status in rat ovary tissues: protective role of black grape extract.

OBJECTIVES: It has been demonstrated that immunosuppressive drug cyclosporine A (CsA) increases the synthesis of reactive oxygen species (ROS) and lipid peroxidation products in vitro and in vivo studies, but its possible toxic effects in ovary tissue have not been defined yet. This study was designed to elucidate possible relation between CsA treatment and oxidant/antioxidant status in rat ovary tissue and, to determine if antioxidant supplementation is beneficial. METHODS: CsA and black grape extracts were given to rats orally for 10 days. In each group (control, CsA, CsA plus black grape and black grape alone), there were 7 animals (28 rats in total). After the animals were sacrificed, their ovaries were removed to be used in the measurement of malondialdehyde (MDA) level and activities of superoxide dismutase (SOD), catalase (CAT) and xanthine oxidase (XO) enzymes. RESULTS: In the control, CsA plus black grape and black grape alone groups, MDA levels were significantly lower than CsA group. Xanthine oxidase activities were significantly higher in CsA and CsA plus black grape groups than control group. In addition, XO activity was significantly lower in black grape alone group than all the other groups including control group. There were no significant differences in the activities of SOD and CAT enzymes between the groups. CONCLUSION: The results suggest that CsA leads to oxidant stress and peroxidation in rat ovary tissues mainly due to increased XO activity. It has been suggested that this oxidant stress and peroxidation reactions can be prevented by antioxidant food supplementation like black grape.

Animals↗
PubMed2006

Effect of in vitro gastric and duodenal digestion on the allergenicity of grape lipid transfer protein.

BACKGROUND: Severe grape allergy has been linked to lipid transfer protein (LTP) sensitization. LTPs are known to be resistant to pepsin digestion, although the effect of gastroduodenal digestion on its allergenicity has not been reported. OBJECTIVE: We sought to investigate the effect of gastric and gastroduodenal digestion on the allergenic activity of grape LTP. METHODS: The proteolytic stability of grape LTP was investigated by using an in vitro model of gastrointestinal digestion. The allergenicity of LTP and its digesta was assessed in vitro by means of IgE immunoblotting, RASTs, and in vivo skin prick tests in the same patients with grape allergy. RESULTS: Grape LTP was resistant to gastric digestion, and yielded a 6000-d relative molecular mass C-terminally trimmed fragment after duodenal digestion. This fragment retained the in vitro IgE reactivity of the intact protein. Inclusion of phosphatidylcholine during gastric digestion protected the LTP to a limited extent against digestion. Digestion did not affect the in vivo (skin prick test) biologic activity of LTP. CONCLUSION: The allergenic activity of grape LTP was highly resistant to in vitro digestion. This property might facilitate sensitization through the gastrointestinal tract and might also potentiate the ability of LTPs to elicit severe allergic reactions in sensitized individuals. CLINICAL IMPLICATIONS: Purified natural allergens will facilitate the development of component-resolved diagnostic approaches, including allergen chips. This study contributes to our understanding of the role digestion plays in symptom elicitation in true food allergy.

Allergens↗
PubMed2004

Influence of several Styrian wines on bovine coronary artery contractions induced by endogenous agents.

In the current study, seven Styrian white wine varieties, as well as their corresponding grape skin extracts (GSE), were tested for possible antagonistic effects on several endogenous vasoconstrictors known to be involved in the pathogenesis of cardiovascular diseases via tissue contraction experiments. Results were compared to those of Zweigelt, the most cultivated Styrian red wine. Bovine coronary artery strips were attached to a force transducer connected to a bridge amplifier, and isometric force was recorded on a multipen recorder. Preincubation of the vessels with the dealcoholized wines (DAW; 330 microl/ml) inhibited the constrictions to histamine and serotonin (5-HT) NO-dependently in the range of 5-80% and 30-90%, respectively (Zweigelt 47% and 90%, respectively). The corresponding GSE (20 mg/ml) inhibited the coronary constrictions to histamine in the range of 40% to 80% (Zweigelt 80%). Additionally, all DAW antagonized the contractile responses to the thromboxane-mimetic U46619 and the isoprostane 8-iso-PGF2alpha, indicating a nonspecific inhibition. To characterize the vasoactive component(s), the Traminer GSE was separated representatively using ultrafiltration, solid phase extraction (SPE) on Isolute C18(EC), and Isolute SCX-2, as well as column chromatography (CC) on polyamide. The resulting fractions were subsequently bioassayed for vasorelaxing activity. Preliminary results refer to a very hydrophilic, nonpolyphenolic basic compound with a MW<1000 Da. Our findings demonstrate that certain Styrian wines have remarkable antagonistic effects on several endogenous agonists in vitro, and that also nonpolyphenolic components in grapes may contribute to cardiovascular protection. Further separation steps as well as phytochemical and pharmacological investigations are in progress.

Animals↗
PubMed2006

Discrimination of Portuguese grapevines based on microsatellite markers.

A set of 46 grapevine denominations was genotyped at 11 microsatellite loci in order to discriminate them. Ninety four alleles with a mean number of 8.55 alleles per locus were observed in a total of 37 detected unique genotypes. Previously assumed synonyms were confirmed and several cases of homonymy resolved. Comparison of the data obtained in this study with data of 32 genotypes previously reported enabled the detection of three parent offspring relationships, and identified other putative parent/progeny relationships. These data allowed understanding the origin of some Portuguese cultivars. The integration of the obtained data with ampelographic data would be very important for the accurate identification of the Portuguese cultivars and can become a significant tool for the certification of quality wines produced in specific regions.

Agriculture↗
PubMed2006

Assay of ochratoxin A in grape by high-pressure liquid chromatography coupled on line with an ESI-mass spectrometry.

In this paper, we propose a method for detection of ochratoxin A (OTA) in grapes by using nano-reversed-phase high-performance liquid chromatography-electrospray ionization-mass spectrometry (nano-RP-HPLC-ESI-MS). The method is rapid, highly sensitive and reproducible. OTA is extracted preferably from the entire acinus, rather than must; using chloroform at long incubation time period, lyophilized, resolubilized in acetonitrile (AcCN) and injected onto a reversed phase capillary or analytical column. Capillary columns are the method of choice because it requires a reduced amount of injected sample and consequently the chloroform necessary for OTA extraction, which is a toxic agent. This method gives a detection limit of femtog/ml, without resorting to an immunoaffinity clean-up or concentration, which makes it by far superior to any other method reported. Moreover, by using MS as a detection method it is possible, in the case of a complex matrix, to measure its molecular mass and to confirm the presence of OTA by MS-MS, which cannot be done by fluorescent detection. The method has a high sample extraction throughput (24/h) and has adequate precision (between batch C.V. <8%) and sensitivity (limit of detection (LOD)=1 pg/g; limits of quantification (LOQ)=2 pg/g) for OTA measured.

Chromatography, Affinity↗
PubMed2005

Removal of lead(II) and cadmium(II) from aqueous solutions using grape stalk waste.

The sorption of lead and cadmium from aqueous solutions by grape stalk waste (a by-product of wine production) was investigated. The effects of the contact time, pH of the solution, ionic medium, initial metal concentration, other metal ions present and ligands were studied in batch experiments at 20 degrees C. Maximum sorption for both metals was found to occur at an initial pH of around 5.5. The equilibrium process was described well by the Langmuir isotherm model, with maximum grape stalk sorption capacities of 0.241 and 0.248 mmol g(-1) for Pb(II) and Cd(II), respectively, at pH around 5.5. Kinetic studies showed good correlation coefficients for a pseudo-second-order kinetic model. The presence of NaCl and NaClO(4) in the solution caused a reduction in Pb and Cd sorption, the latter being more strongly suppressed. The presence of other metals in the uptake process did not affect the removal of Pb, while the Cd uptake was much reduced. HCl or EDTA solutions were able to desorb lead from the grape stalks completely, while an approximately 65% desorption yield was obtained for cadmium. From the results obtained it seems that other mechanisms, such as surface complexation and electrostatic interactions, must be involved in the metal sorption in addition to ion exchange.

Adsorption↗
PubMed2005

Physiological responses of Tunisian grapevine varieties to bicarbonate-induced iron deficiency.

Plants are frequently submitted to iron deficiency when growing on calcareous soils, which contain high concentrations of bicarbonate. The purpose of this study was to investigate the variability of physiological responses of Tunisian grapevine varieties to bicarbonate-induced iron chlorosis. Vine woodcuttings of seven autochthonous Tunisian varieties (Khamri, Mahdaoui, Blan3, Saouadi, Arich Dressé, Beldi and Balta4), two rootstocks (140Ru and S.O.4), and an introduced table variety (Cardinal) were cultivated on inert sand for 2 months using a complete nutrient solution (20 microM Fe) that was either well supplied or not supplied with 10 mM HCO3-. Young leaves of plants cultivated on bicarbonate-enriched medium showed characteristic symptoms of iron chlorosis, although the intensity of the symptoms depended on the variety and the rootstock. Chlorosis score confirmed these observations since the most sensitive varieties showed the highest values. This variability in tolerance to iron deficiency was also displayed when analysing the physiological parameters (shoot length, plant dry weight, and chlorophyll concentration) and the iron contents in the 4th leaf. Analysis of morphological and physiological parameters showed three behaviour groups. The first one corresponded to tolerant varieties (Khamri, Mahdaoui, and the root-stock: 140Ru), the second included moderately tolerant vines (Saouadi, Arich Dressé, Blanc3, and the rootstock: S.O.4) and the third represented the sensitive ones (Balta4, Beldi, and Cardinal).

Bicarbonates↗
PubMed2006

Expression of three expansin genes during development and maturation of Kyoho grape berries.

Expansins are cell-wall-localized proteins that induce loosening of isolated plant cell walls in vitro in a pH-dependent manner, but exhibit no detectable hydrolase or transglycosylase activity. Three putative expansin cDNAs, Vlexp1, Vlexp2, and Vlexp3 were isolated from a cDNA library made from mature berries of the Kyoho grape. Expression profiles of the 3 genes were analyzed throughout berry development. Accumulation of the Vlexp3 transcript was closely correlated with berry softening, and expression of this gene was detected before véraison and markedly increased at véraison (onset of berry softening). Expression of Vlexp3 was berry-specific. Vlexp1 and Vlexp2 mRNA accumulation began during the expansion stage of berry development and expression increased for both genes during ripening. Vlexp1 and Vlexp2 mRNA was detected in leaf, tendril and flower tissues and Vlexp2 mRNA was additionally detected in root and seed tissues. These findings suggest that the three expansin genes are associated with cell division or expansion and berry ripening. Vlexp3, in particular, is most likely to play a role in grape berry softening at véraison.

Amino Acid Sequence↗
PubMed2004

Isolation of recombinant antibodies (scFvs) to grapevine virus B.

A panel of 15 recombinant single chain antibodies (scFv) specific to grapevine virus B (GVB) were recovered from a human combinatorial scFv antibody library using the phage display technique against purified virus particles. Two selected scFv-encoding genes were expressed in recombinant Escherichia coli cells as dimeric antibodies. Successful detection of GVB in tissues of herbaceous hosts and grapevine was obtained in a direct binding assay using dimeric scFvs. This reagent was also shown to substitute efficiently for a GVB polyclonal serum in standard DAS-ELISA test used routinely for diagnosis.

Antibodies, Viral↗